1. Field of the Invention
This invention is generally related to blood analysis instrumentation and, more particularly, to instrumentation and techniques for monitoring the platelet function and clot structure of a blood sample during both clotting and dissolution of clots. In addition, the invention provides a new method of assessing erythrocyte flexibility.
2. Description of the Prior Art
U.S. Pat. No. 4,986,964 to Carr et al. discloses a clot retractometer which measures force development during platelet mediated clot retraction. This instrument has been shown to be a novel gauge of platelet function (see, Carr et al., Blood Coagulation and Fibrinolysis, 2:30-308 (1991), Carr et al., Am. J. of Med. Sci., 302:13-18 (1991), and Carr et al., Blood, 78:482A (1991)). However, clot retraction is dependent on intact platelet membrane structure, normal platelet metabolic function, fibrin structure and normal platelet-fibrin interactions. Changes in clot retraction are sensitive to a spectrum of both fluid phase and platelet abnormalities. Force development is completely dependent on platelet function and if platelet function is abnormal or if no platelets are present, force development will be completely absent.
When tissue injury occurs, bleeding is halted and vascular integrity is restored by activation of the hemostatic mechanism. A complex combination of blood protein interactions and cellular activations leads first to the formation of a platelet plug and subsequently to the production of the fibrin-platelet clot. Since the amount of blood loss is directly proportional to the time required to stop the bleeding, both sets of reactions occur rapidly. While the ability to stop injury-induced blood loss is critical, the capability of turning flowing blood into a solid is a source of serious problems. Clot formation within a vessel leads to decreased flow, ischemic damage and, if the clot is not removed, eventual tissue infarction.
To avoid clot formation in non-injured tissue and to prevent clot propagation from the site of injury to other locations within the vascular bed, the coagulation system is balanced by a series of potent inhibitors. Furthermore, the clot itself is designed as a temporary patch. Once bleeding stops and tissue repair is initiated, the clot is dissolved by the enzyme plasmin.
Under normal conditions, the coagulation system remains in a fine balance. Pathologic alterations of the system may induce a risk of hemorrhage or increase the potential for thrombosis. An example of the former would be the bleeding disorder of hemophilia which results from a low level of Factor VIII, a blood clotting protein. An example of the latter would be recurrent venous thrombosis in individuals who have decreased levels of the coagulation inhibitor antithrombin III. Patients with decreased ability to remove clots, decreased fibrinolytic potential, are also at risk for thrombosis.
Currently, the evaluation of patients for increased risk of bleeding is accomplished through a series of coagulation screening tests. The prothrombin time (PT) and partial thromboplastin time (PTT) identify patients at risk for bleeding and direct the clinician to more specific tests to define the cause of the increased risk. If the PT is prolonged (i.e. takes longer to clot than normal plasma), the patient is at increased risk for bleeding. Since prolongations of the PT are known to occur in specific factor deficiencies, the appropriate factor levels can be measured to define the abnormality. Unfortunately, screening tests for fibrinolytic potential are presently not available. Furthermore, tests which identify patients at risk for thrombosis due to decreased ability to dissolve clots have not been reported and are not in use.
Clot dissolution can be monitored using clot optical density measurements or by measurement of radioactive material release from the clot. The optical density technique relies on the fact that clot formation increases the turbidity of the plasma sample while clot dissolution reduces turbidity back to baseline, pre-clot values (see, Carr et al., Thromb. Haemostas., 67:106-110 (1992)). In this technique, clotting is monitored as a rise in turbidity, while clot dissolution is seen as a fall in clot turbidity. The radioactive tracer technique involves the addition of radiolabelled clotting protein, for example .sup.125 I labelled fibrinogen, to the patient sample (see, Carr et al., Thromb. Haemostas., 67:106-110 (1992) and Knight et al., Thromb. Haemostas., 46:593-596 (1981)). As clotting occurs, the labelled fibrinogen is incorporated into the clot structure. During subsequent clot dissolution, radioactive fragments are released. The rate of radioactivity release is proportional to the rate of clot dissolution.
A major problem with the turbidity technique is that it cannot be utilized in systems containing erythrocytes. A major problem with the tagged fibrinogen technique is the necessity for using radioactive material. Moreover, a divergence in results has been observed with the two techniques. When dissolution is rapid, both techniques yield comparable dissolution times. However, when dissolution is delayed, dissolution times measured by the turbidity technique tend to be longer than those measured by .sup.125 I release (see, Carr et al., Thromb. Haemostas., 67:106-110 (1992)).
Altered erythrocyte deformability is thought to play a role in multiple disease processes. Unfortunately, measurement of erythrocyte flexibility remains somewhat problematic. Currently, the majority of investigators have utilized erythrocyte filtration techniques to measure flexibility. The common feature of these methods is the flowing of erythrocytes through filters which typically have pores of uniform size. Erythrocytes either flow through under low pressure (e.g., a static column of blood), or are forced through at higher pressures. Results are reported as a "filtration index". Filtration techniques have several major deficiencies. First, they are difficult to reproduce. Results vary from laboratory to laboratory, and may vary over time in the same laboratory. Second, they fail to take into account the possibility that microvascular channels may be flexible. Third, they ignore possible contributions of the clotting system. Another prior method of measuring erythrocyte flexibility is called ektocytometry. This method involves the deformation of cells in shear fields with simultaneous monitoring of shape change. Ektocytometry has the advantage of being a non-flow method of monitoring erythrocyte flexibility; however, it is indirect, expensive, and not widely available. In view of the above, it would be advantageous to have an inexpensive and reproducible method of evaluating erythrocyte flexibility.